Project Summary Melanoma is an aggressive skin tumor arising from melanocytes. While it is well documented that cell heterogeneity is prevalent within melanomas with cells varying widely in their degree of pigmentation and cell morphology, clinical treatment of melanoma does not take into account these diverse differentiation states. The mechanisms underlying this phenotypic heterogeneity remain poorly understood, but play important role in drug sensitivity and metastatic capacity. One determinant of phenotypic heterogeneity is the differentiation state of the cell, which can be due to both cell-intrinsic and microenvironmental factors. We have identified a series of genes that play a role in melanoma differentiation state, including the sheddase BACE2. In Aim 1, we demonstrated that human melanomas strongly overexpress BACE2. Using a zebrafish BACE2-/- mutant zebrafish, we showed that BACE2 loss of function leads to enforced differentiation of melanocytes. This suggests that BACE2 is a differentiation gatekeeper that modulates the proper balance between neural crest and melanocyte states. In Aim 2, we will investigate the impact of differentiation on melanoma by manipulating BACE2 level with an emphasis on cell proliferation and metastasis. We will utilize zebrafish transplantation and transgenic models to dissect the step-wise influence of differentiation on primary tumor growth and metastasis. We will then extend this work to human cancer by engineering melanoma cell lines with inducible knockdown of BACE2. These studies will shed light on how BACE2, a gene involved in melanocyte differentiation, affects melanoma growth and metastasis. In my predoctoral training, I will gain a deeper understanding of the tumor heterogeneity resulting from cell intrinsic differentiation status, and this training will prepare me for my transition into postdoctoral research with a focus on how the extrinsic tumor microenvironment shapes tumor heterogeneity.